PROJECT SUMMARY/ABSTRACT Qualitative changes in the American diet have been linked to the development of type 2 diabetes mellitus (T2DM). An essential component in the development of T2DM is impaired ?-cell glucose-stimulated insulin secretion (GSIS). GSIS is augmented by saturated long-chain fatty acids (FA), but is impaired by both dietary and infused ?-6 polyunsaturated FAs (PUFA). Intracellular metabolism of FA begins with its thioesterification by acyl-CoA synthetase (ACSL) with coenzyme A (CoA) to produce an acyl-CoA. We hypothesize that ?-6 PUFA impairs GSIS and disrupts glucose homeostasis by reducing ?-cell ACSL isoform 4 (ACSL4) expression and increasing levels of unesterified epoxyeicosatrienoic acids (EETs). This hypothesis is strongly supported by several observations: i) exposing INS 832/13 insulinoma ?-cells to ?-6 PUFAs (arachidonate or linoleate) not only impairs GSIS, but also reduces ACSL4 mRNA and protein expression; ii) incubating INS 832/13 cells with unesterified EETs decreases GSIS by 30%; iii) ACSL4 activates EETs to form EET-CoAs, which are incorporated into glycerolipids, thereby preventing unesterified EETs from accumulating and impairing GSIS; and iv) mice that are deficient in ?-cell-specific ACSL4 are hypoinsulinemic and hyperglycemic. In this proposal, we will test the specific hypothesis that unesterified EETs impair GSIS and that ACSL4 activity is essential in preventing the accumulation of unesterified EETs. In Aim 1, we will determine the mechanism by which unesterified EETs impair GSIS by evaluating how EETs affect steps in the exocytosis of insulin. In Aim 2, we will determine how dietary ?-6 PUFA increase intracellular unesterified EETs by determining how ?-6 PUFAs regulate ACSL4 expression and activity. In Aim 3, we will determine the role of ?-cell ACSL4 in maintaining whole body glucose homeostasis in the setting of different high FA diets. Using mouse pancreatic islets, ?-cell lines, and animal models, this proposal will examine unique and previously unexplored pathways in the regulation in insulin secretion. The control of insulin exocytosis by EETs is entirely novel, and should lead to new understanding of insulin insufficiency in T2DM. Additionally, our ?-cell-specific ACSL4 knockout mouse provides a novel model for T2DM that mimics the insulin secretion defect that is present in the human disorder. The expected outcome of this proposal is a greater knowledge of how dietary FA impact insulin secretion and whole body glucose homeostasis. By investigating the metabolism of EETs in ?-cells, critical data will be obtained on the insulin secretory pathway and its regulation by different dietary FAs. Understanding these mechanisms will enable us to develop targeted dietary therapies to prevent and treat T2DM.